Utilization of Medicinal Plants and Their Products in the Treatment and

Chapter 7

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Utilization of Medicinal Plants and Their Products in the Treatment and Control of Disease in Fish Albert P. Ekanem* and Austin I. Obiekezie Institute of Oceanography, University of Calabar, Calabar, Nigeria *E-mail: [email protected].

African medicinal plants and their products have shown efficacies in the treatment of diseases in farmed fish. Extracts of Carica papaya and Mucuna pruriens were effective in the treatment of Ichthyophthiriasis in goldfish (Carassius auratus auratus) with high host tolerance. Similarly, ectoparasitic monogeneans were effectively dislodged from the gills and skin of goldfish by the application of extracts of Piper guineense at therapeutic concentrations. Artemesia annua with known antimalarial activities in human was also effective against fish monogenean parasites of Clarias gariepinus in a concentration related manner without detriments to the host. Effective antibacterial activities against Aeromonas and Pseudomonas diseases of the African catfish (Heterobranchus longifilis) were demonstrated in some plants such as Phylanthus amarus, Allium sativum, A. annua, Citrus lemon with minimum inhibitory concentration (MIC) between 25 to 100mg/ml. African plants have shown potential promise in the treatment of diseases in fish and other aquatic animals.

Aquaculture Development in Nigeria The purpose of aquaculture production is to increase the availability of animal protein through fish culture with the aims of making it available to as many people as possible. Presently, the high cost of animal flesh (cow, goat, pork, ram, chicken etc) has made it inaccessible to the common man in some developing countries. © 2013 American Chemical Society In African Natural Plant Products Volume II: Discoveries and Challenges in Chemistry, Health, and Nutrition; Juliani, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2013.


Moreover, some people, because of religious and cultural affiliations, are critical about the consumption of flesh from some animals, but fish is the only animal protein source that has bridged cultural and religious affiliations, being widely consumed. It is the cheapest source of animal protein in Nigeria and other African countries, commonly found in most people’s meal. On the estimate, more than 98% of the fish consumed in Nigeria are cropped from the wild and this stock of wild fish is gradually diminishing due to over fishing. Aquaculture production presents the only alternative means to the wild stock of fish to meet the demand of the increasing Nigerian population. Aquaculture practice in the Sub-Saharan African countries has been crippled by several factors prominent among which are economic losses from fish mortalities due to outbreak of diseases and lack of affordable drug for their controls and management. Presently, and unlike other agricultural practices, most of the available drugs for the treatment of aquaculture diseases are imported from Europe and America, thereby making the practice very expensive for an average African farmer. Most of the farmers who have tried their hands on fish production are making use of natural stream waters with highly degraded quality, thereby predisposing their candidates (fish) to diseases. The commonest disease problems encountered are parasitic and bacterial and a few cases of fungal related cases as secondary infections. The most frequent parasitic problems associated with tropical aquaculture include trichodiniasis and monogeneasis. The bacteria associated with fish mortalities are mostly gram negative organisms, the Pseudomonas, Aeromonas and Flexibacter species. These organisms poses enormous problem to hatchery raised fish juveniles and brood fish, resulting in economic losses and abandonment of projects by some farmers. The research on African medicinal plants was informed by their availability and also by the fact that they have been found effective for the treatment of diseases in human and other animals of veterinary importance. It has long been recognized that naturally occurring substances in higher plants have antibacterial activities (1). The use of medicinal plants and their products in the treatment of diseases is a widespread practice (2). Currently there is an increasing demand for medicinal plants and plant products as alternative to orthodox medicines especially in developing countries (3). It is also important to develop a therapeutic source that will have little or no impact on the environment, being informed by the deleterious impact on the environment by the use of chemical-based substances for the treatment of aquaculture diseases in the developed countries of the world. Moreover, the problems associated with drug accumulation in the tissues of food fish can be solved or reduced by the use of plants and their products in the management of disease problems. There are however, relatively few publications on the ethno medical and ethno botanical uses of plants or their products in the treatment of diseases in aquaculture. Large numbers of plants are rapidly becoming extinct and need to be urgently investigated for their medicinal properties. Plant-based substances and other natural products have been extensively used for the treatment of human diseases, ailments and partly for the treatment of diseases in other animals excluding fish. 94

In African Natural Plant Products Volume II: Discoveries and Challenges in Chemistry, Health, and Nutrition; Juliani, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2013.


The need for the development of alternative methods of treatment of aquaculture diseases is informed by the fact that some of the effective chemical-based substances which are presently used (Table I) for the treatments of aquaculture diseases are not recommended for food fish. A typical example is malachite green which is effective for the treatment of protozoan infection in cultured fish but a potential mutagen in human consuming such products (fish) and highly restricted from used in some countries (4). Moreover, the continuous use of chemical substances in the aquatic environment is likely to impact negatively on the environment. The review on the utilization of Medicinal plants and their products in the treatment of fish diseases is designed to provide adequate information on their efficacies, toxicities (Tables II, III and IV) and pharmacological active principles as recommended in the African pharmacopoeia by the World Health Organization. The use of medicinal plants and their products in the treatment of diseases is a widespread practice (2). Currently there is an increasing demand for medicinal plants and plant products as alternative to orthodox medicines especially in developing countries (3).

Table I. Mortality of Goldfish Heavily Infested with Ichthyopthirius multifiliis after Bath Treatment with Petroleum Ether Extract of Carica papaya and Methanolic Extract of Mucuna pruriens for 96 h Concentration (mg/l)

Fish Mortality (%) Carica papaya

Mucuna pruriens

0 (Control)

48

65

100

40

50

150

30

30

200

15

20

Efficacies of Extracts of Mucuna pruriens and Carica papaya Against Ichthyophthirius multifiliis of Goldfish The ciliate Ichthyophthirius multifiliis is among the most pathogenic parasites of fish maintained in captivity. According to Ekanem et al (5), the effects of the crude methanolic extract of leaves of Mucuna pruriens and the petroleum-ether extract of seeds of Carica papaya against I. multifiliis were investigated under in vivo and in vitro conditions. Goldfish (Carassius auratus) infected with the parasites were immersed for 72 h in baths with M. pruriens extract, and for 96 h in baths with C. papaya extract. There was a 90% reduction in numbers of I. multifiliis on fish after treatment in baths of each plant extract at 200 mg/l compared 95 In African Natural Plant Products Volume II: Discoveries and Challenges in Chemistry, Health, and Nutrition; Juliani, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2013.

to untreated controls. Consequently, parasite-induced fish mortality was reduced significantly. A complete interruption of trophont recruitment was achieved by immersion in the M. pruriens extract. In vitro tests led to a 100% mortality of I. multifiliis in 150 mg/l M. pruriens extract, and in 200 mg/l of C. papaya extract after 6 h. Although the active constituents of the medicinal plant extracts are still unknown, it was demonstrated that M. pruriens and C. Papaya have potentials for effective control of I. multifiliis.


Efficacy of Extracts of Piper Guineense Schum. and Thonn. against Skin And Gill Monogenean Parasites of Fish under in Vivo and in Vitro Conditions Monogenea is one of the most economically important parasites of cultured fish (6). The viviparous Gyrodactylids parasitizes the skin, fin and gills of freshwater and marine fishes, whereas the oviparous Dactylogyrids are mainly gill parasites of freshwater fish (7). Two of the most important species of fish monogeneans are Dactylogyrus extensus and Dactylogyrus vastator (8), which are endemic in Asia, Central Europe, Middle East and North America (9).

Figure 1. Effect of a 96 h bath treatment with petroleum ether extract of Carica papaya on Ichthyophthirius multifiliis on the skin of goldfish. Given are the mean numbers and standard deviations of juvenile and mature trophonts on the skin (including fins). Asterisk indicate significantly decreased parasite numbers (p ≤ 0.05) compared to the lower concentrations and control, respectively. 96 In African Natural Plant Products Volume II: Discoveries and Challenges in Chemistry, Health, and Nutrition; Juliani, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2013.


According to Ekanem et al (10), methanol extracts of the seeds of Piper guineense (Piperaceae) were active against goldfish (Carassius auratus auratus L. Pisces Cyprinidae) monogenean parasites. The seed extract of P. guineense was administered at different concentrations of 0.5–2.0 mg/L, under in vivo and in vitro conditions. There was a higher efficacy of the effects of the extracts against fish parasites under in vitro situations than under in vivo. According to the report, There was a positive correlation (Fig. 1) between goldfish (Carassius auratus auratus) parasite (Gyradactylus and Dactylogyrus) mortalities and the concentration of methanol extracts of Piper guineense. An equal percentage fish mortality was observed in the extract concentrations 0.5, 1.0 and 1.5 mg/L, but later increased with increasing natural product concentration (Fig. 2). There were more parasites on the skin of goldfish than on the gills. Considering the fish and parasite mortalities between the control and the test groups, a marked reduction of fish mortalities was observed in the control group compared with fish mortalities in the treated groups, which increased with increasing extract concentration. There was zero parasite mortality on both the skin and gills of the fish in the control groups.

Figure 2. Effect of a 96 h bath treatment with methanolic extract of Mucuna pruriens on Ichthyophthirius multifiliis on the gills of goldfish. Given are the mean numbers and standard deviations of juvenile and mature trophonts on the gills. Asterisk indicate significantly decreased parasite numbers (p≤0.05) compared to the lower concentrations and control, respectively.

A higher efficacy against fish parasites in the in vitro tests was observed in the treated groups when the extracts were at a concentration of 1.5 and 2.0 mg/L. There was a 60% and 80% reduction of skin and gill parasites in 1.5 mg/L extract, 97 In African Natural Plant Products Volume II: Discoveries and Challenges in Chemistry, Health, and Nutrition; Juliani, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2013.

whereas a 70% and 100% parasite reduction on the skin and gill respectively, were observed in 2.0 mg/L seed extracts of P. guineense. Comparing results from the in vivo and in vitro screens, anthelmintic activity was observed earlier from the extract in the in vitro screens than in the in vivo.


Efficacy of Ethanol Extracts of Artemisia Annua L. Against Monogenean Fish Parasites Of Heterobranchus Longifilis Artemisia annua L., also known as annual wormwood or Sweet Annie, belongs to the family Asteraceae. It is a highly aromatic herbaceous plant of Asiatic and Eastern European origin, widely dispersed throughout the temperate region (11, 12). It was used by Chinese herbalists since A.D. 341 for the treatment of fevers associated with malaria (13). Its activity against malarial parasites in primate models was demonstrated in 1971, but the isolation and characterization of the active antimalarial principle, artemisinin, by Chinese scientists were in 1972 (14).

Figure 3. Parasite mortality against concentrations (mg/l) of Artemisia annua at different time intervals. According to Ekanem and Brisibe (15), ethanol extract of Artemisia annua was effective in the dislodgement and mortality of monogenean parasites of juvenile Heterobranchus longifilis at concentrations ranging from 50 to 200 mg/l. Five hundred 1-week-old juvenile fish were stocked in hapa in earthen pond for 7 days to accumulate parasites. The approximate number of parasites per fish was confirmed by counting the number of parasites attached to body surfaces and the gills with a stereo-microscope before being exposed to the extract under in vivo conditions. The bioactivity of the extract was conducted in plastic Petri dishes with three replications and controls. The results obtained from A. annua extract (Fig. 3) were matched against those produced by pure artemisinin (Fig. 4) and artesunate powder (Fig. 5), respectively, under similar experimental conditions. There was a faster effect of pure artemisinin crystals on the parasites as compared 98



to A. annua extract and artesunate. Coagulation of parasite cells was observed with artemisinin treatment, whereas parasites were merely dislodged from their attachment organs and killed some hours later in the same concentration of A. annua. There were positive correlations between the number of parasites dislodged/killed and the concentration of A. annua extract, artemisinin, and artesunate powder, respectively, as well as the duration of exposure of affected fish to the substances. This led to the conclusion that A. annua contains substances that are effective against helminthes parasites of H. longifilis.

Figure 4. Parasite mortality against concentrations (mg/l) of Artemisin at different time intervals.

Figure 5. Parasite mortality against concentrations (mg/l) of Artesunate at different time intervals. 99 In African Natural Plant Products Volume II: Discoveries and Challenges in Chemistry, Health, and Nutrition; Juliani, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2013.


Examination of the fish fry at the end of the test periods demonstrated that while some of the fish that previously harbored parasites were found to be free of some of the parasites, others were completely free of the parasites. Comparatively, the number of parasites on the body surfaces of fish in the control was the same throughout the test period. The concentration of A. annua in which 50% of the parasites were killed was 100 mg/l within 60 minutes and a significant number (about 85%) were killed in 200 mg/l. In treatment with artemisinin, 50% of the parasites were killed in 70 mg/l after exposure for 30 minutes while all parasites were killed with their cells coagulated in 100 mg/l within 60 minutes of exposure. Fifty percent parasite mortality was observed in 85 mg/l of Artesunate after exposure for 90 minutes. A mortality rate of about 85% of the parasites occurred when the fish fry were bathed in solution containing 150 mg/l of artesunate for 90 minutes. Interestingly, monogenean parasites were all dislodged from their attachment sites before the occurrence of mortality following treatment with artesunate.

Table II. Toxicity Test of Concentrations (mg/l) of A. annua Against Fish Mortality (%) Conc. Mg/l

Fish Mortality (%) 24H

48H

72H

96H

250

0

10

0

10

300

0

10

10

10

350

10

20

10

10

400

10

10

10

20

500

20

10

20

20

It was also observed that the parasite loads were reduced with increasing concentrations of A. annua extract. There was a positive correlation between the number of parasites dislodged from the body surfaces of fish and the time of exposure of fish to the extract. In addition to all of these, an increased agility in the swimming of fish freed from parasites was also observed when compared to their counterparts in the control with all parasites remaining intact. Results of the toxicity test showed that extract of A. annua was well tolerated by H. longifilis juveniles. A minimal mortality observation was made throughout the 96 h period of exposure of fish to the extract. A few fish showed weak swimming activity in 350 to 500 mg/l of the test solutions. The highest percentage mortality observed after 96 h in the highest concentration was 20%. 100 In African Natural Plant Products Volume II: Discoveries and Challenges in Chemistry, Health, and Nutrition; Juliani, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2013.


Table III. Showing Host (Fish) Tolerance to Effective Plants Against Fish Ectoparasites Plant extract

Mode of application

Curative dose (EC50)

Threshold conc. (mg/l)

Lethal dose (96 h LC50)

Therapeutic index (TI)= (LC50/EC50)

Remarks

Mucuna pruriens

Long bath in water

200 mg/l

1000

1200 mg/l

6

High host tolerance

Carica papaya

Long bath in water

200 mg/l

1200

1600 mg/l

8

High host tolerance

1 mg/l

1.5

2 mg/l

2

Indication of host toxicity

Piper Long bath guineense in water

Table IV. Standard Threshold Concentrations of Substances and Their Toxicity Gradients to Aquatic Organisms (16) Group

Threshold concentration (mg/l)

A

> 500

Hardly toxic

B

100-500

Weakly toxic

C

10-99

D

1-9

Considerably toxic

E

< 1

Highly toxic

Toxicity grade

Moderately toxic

References 1. 2. 3. 4.

5.

Cha, J. D.; Jeong, M. R.; Choi, H. J.; Jeong, S. I.; Moon, S. E.; Yun, S. I.; Kim, Y. H.; Kil, B. S.; Song, Y. A. Planta Med. 2005, 71, 575–577. Iwu, M. M. Ethnomed. 1982, 2 (1-4), 45. Murray, N. D. African Indigenous Medicinal, African Medicinal and Research Function; Nairobi, Kenya, 1998; pp 37−38. Commiittee on Mutagenic Chemicals in Food, Consumer Products and the Environment. Statement for COT: Malachite Green and Leucomalachite Green; Department of Health: London, 1999. Ekanem, A. P.; Obiekezie, A.; Kloas, W.; Knopf, K. Parasitol Res. 2004, 92, 361–366. 101


6. 7. 8. 9. 10. 11.


12.

13. 14. 15. 16.

Wooten, R. In Fish Pathology; Roberts, R. J., Ed.; Bailliere-Tindall: London, 1989; pp 228–242. Yamaguti, S. Monogenetic Trematodes of Hawaiian Fishes; University of Hawaii Press: Honolulu, HI, 1968, 287. Musselius, V. A. In Investigations of Monogeneans in the USSR; Skarlato, O. A., Ed.; Oxonian: New Delhi, 1987. Paperna, I. J. Parasitol 1964, 50, 90–93. Ekanem, A. P.; Wang, M.; Simon, J. E.; Obiekezie, A. I.; Morah, F. Phytother. Res. 2004, 18, 793–797. Bailey, L. H.; Bailey, E. Z. Hortus Third; MacMillan Publ. Co.: NewYork, 1976. Simon, J. E.; Chadwick, A. F.; Craker, L. E. Herbs: An Indexed Bibliography, 1971-1980. The Scientific Literature on Selected Herbs, and Aromatic and Medicinal Plants of the Temperate Zone; Archon Books: Hamden, 1984. Hien, T. T.; White, N. J. Lancet 1993, 341, 603–608. Yeung, H.-C. Handbook of Chinese Herbs and Formulas; Institute of Chinese Medicine: Los Angeles, 1985. Ekanem, A. P.; Brisibe, E. B. Parasitol Res. 2010, 106, 1135–1139. Schaperclaus, W. Fish Diseases; Oxonian Press, PVT: New Delhi, 1991; Vol. 2.

102 In African Natural Plant Products Volume II: Discoveries and Challenges in Chemistry, Health, and Nutrition; Juliani, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2013.

Utilization of Medicinal Plants and Their Products in the Treatment and

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